Understanding how stem cells balance self-renewal with differentiation is paramount in harnessing their therapeutic potential in medicine. While recent work has identified master transcriptional regulator genes controlling pluripotency in mouse and human embryonic stem (ES) cells, it is still unclear whether similar mechanisms direct cell fate decisions in tissue-specific unipotent stem cells, such as the spermatogonial stem cells (SSCs) found in mammalian testes. In contrast to the master regulator proteins in ES cells, recent works has discovered essential functions for cell type-specific or -enriched forms of the core transcription machinery in regulating gene expression. These are required to drive critical gene expression programs involved in diverse developmental events associated with multicellularity and organogenesis. The diversification of core transcription machinery is most notable in the germline where germ cell-specific or -enriched variants of the general transcription factors TFIIA and TFIID execute highly critical and selective functions in regulating reproduction and fertility. This new paradigm of germ cell-specific gene regulation is conserved between invertebrates and vertebrates, and may reflect an important mechanism for evolving germ cell-specific modes of gene regulation. However, the need to understand the molecular mechanisms underlying such exquisite regulation of fertility persists. The goal of this research proposal is to characterize the role of TAF4b in the establishment and maintenance of the self-renewing lineage of SSCs in the mouse testis. TAF4b is a gonad-enriched general transcription factor varient and both male and female Taf4b-deficient mice exhibit reproductive defects. Several lines of evidence suggest it plays an essential role in spermatogonia stem cell (SSC) self-renewal and longevity. One of the specific aims in this research proposal is to identify and characterize TAF4b-interacting protein cofactors to investigate their function in SSC regulation. One of the ways TAF4b may control SSC-specific gene expression is through direct interaction with other proteins that function to turn appropriate SSC genes on or off. This aim will test this hypothesis by identifying these proteins and analyzing their function in SSC gene expression. The second specific aim is to identify where TAF4b binds in the SSC genome and identify which genes TAF4b is directly targeting for regulation. This will provide mechanistic insight into how TAF4b determines which genes it will regulate, how SSC-specific gene regulatory programs are established and how these programs drive SSC self-renewal. The work proposed here, to uncover the mechanisms of TAF4b in regulating SSC maintenance and self-renewal, will reveal fundamental biological principles underlying both stem cell biology and reproductive functions in men required for fertility. These studies may reveal the etiologies behind unexplained male infertility and lead to advanced diagnostic and therapeutic tools to better address and manage male infertility.